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Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

Bischof LJ, Kao CY, Los FC, Gonzalez MR, Shen Z, Briggs SP, van der Goot FG, Aroian RV - PLoS Pathog. (2008)

Bottom Line: We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR.The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities.These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

ABSTRACT
Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

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The IRE-1 UPR is activated in response to PFTs.(A) xbp-1 mRNA splicing is induced in wild-type C. elegans fed E. coli expressing Cry5B compared to control E. coli not expressing Cry5B. The time the worms were allowed to feed on the E. coli before total RNA was prepared for RT-PCR is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (B) Compared to worms fed control non-Cry5B expressing E. coli, in vivo activation of hsp-4::GFP occurs specifically in the intestines of worms fed Cry5B expressing E. coli at 20°C for 8 hours. As a comparison for GFP induction, separate worms on control bacteria were heat shocked at 30°C for 8 hours to induce the ER stress response by causing unfolded proteins. The heat shock worms have a strong increase in GFP throughout the body including the head, intestine and hypodermis. Thus, although the entire worm is capable of activating the ire-1-xbp-1 pathway as judged by hsp-4 induction, activation in Cry5B-fed animals is occurring only in those cells targeted by the PFT. Images taken by light microscopy are compared to images with fluorescence microscopy. Scale bar is 0.2 mm. The experiment was performed three times, and representative worms are shown. (C) Aerolysin induces activation of IRE1 in mammalians cells. Exposure of HeLa cells to proaerolysin (2 ng/mL) leads to increased production of spliced XBP1 protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). DTT (10 µg/mL for 2 h) was used as a positive control. Positions of molecular weight markers (kDa) are indicated on right side of the figure. A nonspecific antibody-reacting band was used as a loading control and normalization of the XBP1 signal in each lane.
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ppat-1000176-g001: The IRE-1 UPR is activated in response to PFTs.(A) xbp-1 mRNA splicing is induced in wild-type C. elegans fed E. coli expressing Cry5B compared to control E. coli not expressing Cry5B. The time the worms were allowed to feed on the E. coli before total RNA was prepared for RT-PCR is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (B) Compared to worms fed control non-Cry5B expressing E. coli, in vivo activation of hsp-4::GFP occurs specifically in the intestines of worms fed Cry5B expressing E. coli at 20°C for 8 hours. As a comparison for GFP induction, separate worms on control bacteria were heat shocked at 30°C for 8 hours to induce the ER stress response by causing unfolded proteins. The heat shock worms have a strong increase in GFP throughout the body including the head, intestine and hypodermis. Thus, although the entire worm is capable of activating the ire-1-xbp-1 pathway as judged by hsp-4 induction, activation in Cry5B-fed animals is occurring only in those cells targeted by the PFT. Images taken by light microscopy are compared to images with fluorescence microscopy. Scale bar is 0.2 mm. The experiment was performed three times, and representative worms are shown. (C) Aerolysin induces activation of IRE1 in mammalians cells. Exposure of HeLa cells to proaerolysin (2 ng/mL) leads to increased production of spliced XBP1 protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). DTT (10 µg/mL for 2 h) was used as a positive control. Positions of molecular weight markers (kDa) are indicated on right side of the figure. A nonspecific antibody-reacting band was used as a loading control and normalization of the XBP1 signal in each lane.

Mentions: In a genetic screen for genes involved in the cellular response of C. elegans to the PFT Cry5B, we found a mutant predicted to be defective in protein N-glycosylation in the ER (L.J.B. and R.V.A., manuscript in preparation). Since defects in protein glycosylation induce the UPR, this result suggested that perhaps the UPR might play a role in protection against PFTs. To test this hypothesis, we first investigated whether or not the UPR was activated by a PFT. The xbp-1 gene is spliced upon activation of the IRE-1 branch of the UPR, and its splicing is one marker for IRE-1 (and UPR) activation [13]. In C. elegans, the xbp-1 intron spliced by IRE-1 is 23 nucleotides and the induction of this splicing event can be detected by RT-PCR [14]. To analyze xbp-1 mRNA transcript splicing, animals were fed Escherichia coli expressing Cry5B and compared to worms fed control E. coli (Figure 1A). While there is abundant unspliced xbp-1 mRNA transcript in both samples, there is an increase in the spliced xbp-1 transcript from worms ingesting Cry5B, indicating activation of the IRE-1 pathway. Quantitative analyses indicate that the xbp-1 spliced transcript increases 2.3, 3.0, and 3.0 fold at the 7, 8, and 9 h time points respectively.


Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

Bischof LJ, Kao CY, Los FC, Gonzalez MR, Shen Z, Briggs SP, van der Goot FG, Aroian RV - PLoS Pathog. (2008)

The IRE-1 UPR is activated in response to PFTs.(A) xbp-1 mRNA splicing is induced in wild-type C. elegans fed E. coli expressing Cry5B compared to control E. coli not expressing Cry5B. The time the worms were allowed to feed on the E. coli before total RNA was prepared for RT-PCR is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (B) Compared to worms fed control non-Cry5B expressing E. coli, in vivo activation of hsp-4::GFP occurs specifically in the intestines of worms fed Cry5B expressing E. coli at 20°C for 8 hours. As a comparison for GFP induction, separate worms on control bacteria were heat shocked at 30°C for 8 hours to induce the ER stress response by causing unfolded proteins. The heat shock worms have a strong increase in GFP throughout the body including the head, intestine and hypodermis. Thus, although the entire worm is capable of activating the ire-1-xbp-1 pathway as judged by hsp-4 induction, activation in Cry5B-fed animals is occurring only in those cells targeted by the PFT. Images taken by light microscopy are compared to images with fluorescence microscopy. Scale bar is 0.2 mm. The experiment was performed three times, and representative worms are shown. (C) Aerolysin induces activation of IRE1 in mammalians cells. Exposure of HeLa cells to proaerolysin (2 ng/mL) leads to increased production of spliced XBP1 protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). DTT (10 µg/mL for 2 h) was used as a positive control. Positions of molecular weight markers (kDa) are indicated on right side of the figure. A nonspecific antibody-reacting band was used as a loading control and normalization of the XBP1 signal in each lane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553261&req=5

ppat-1000176-g001: The IRE-1 UPR is activated in response to PFTs.(A) xbp-1 mRNA splicing is induced in wild-type C. elegans fed E. coli expressing Cry5B compared to control E. coli not expressing Cry5B. The time the worms were allowed to feed on the E. coli before total RNA was prepared for RT-PCR is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (B) Compared to worms fed control non-Cry5B expressing E. coli, in vivo activation of hsp-4::GFP occurs specifically in the intestines of worms fed Cry5B expressing E. coli at 20°C for 8 hours. As a comparison for GFP induction, separate worms on control bacteria were heat shocked at 30°C for 8 hours to induce the ER stress response by causing unfolded proteins. The heat shock worms have a strong increase in GFP throughout the body including the head, intestine and hypodermis. Thus, although the entire worm is capable of activating the ire-1-xbp-1 pathway as judged by hsp-4 induction, activation in Cry5B-fed animals is occurring only in those cells targeted by the PFT. Images taken by light microscopy are compared to images with fluorescence microscopy. Scale bar is 0.2 mm. The experiment was performed three times, and representative worms are shown. (C) Aerolysin induces activation of IRE1 in mammalians cells. Exposure of HeLa cells to proaerolysin (2 ng/mL) leads to increased production of spliced XBP1 protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). DTT (10 µg/mL for 2 h) was used as a positive control. Positions of molecular weight markers (kDa) are indicated on right side of the figure. A nonspecific antibody-reacting band was used as a loading control and normalization of the XBP1 signal in each lane.
Mentions: In a genetic screen for genes involved in the cellular response of C. elegans to the PFT Cry5B, we found a mutant predicted to be defective in protein N-glycosylation in the ER (L.J.B. and R.V.A., manuscript in preparation). Since defects in protein glycosylation induce the UPR, this result suggested that perhaps the UPR might play a role in protection against PFTs. To test this hypothesis, we first investigated whether or not the UPR was activated by a PFT. The xbp-1 gene is spliced upon activation of the IRE-1 branch of the UPR, and its splicing is one marker for IRE-1 (and UPR) activation [13]. In C. elegans, the xbp-1 intron spliced by IRE-1 is 23 nucleotides and the induction of this splicing event can be detected by RT-PCR [14]. To analyze xbp-1 mRNA transcript splicing, animals were fed Escherichia coli expressing Cry5B and compared to worms fed control E. coli (Figure 1A). While there is abundant unspliced xbp-1 mRNA transcript in both samples, there is an increase in the spliced xbp-1 transcript from worms ingesting Cry5B, indicating activation of the IRE-1 pathway. Quantitative analyses indicate that the xbp-1 spliced transcript increases 2.3, 3.0, and 3.0 fold at the 7, 8, and 9 h time points respectively.

Bottom Line: We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR.The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities.These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

ABSTRACT
Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

Show MeSH
Related in: MedlinePlus